Journal: Frontiers in Immunology

Article Title: Development and evaluation of an inhalable nanoemulsion system for enhancing NK cell function against osteosarcoma pulmonary metastases

doi: 10.3389/fimmu.2026.1772375

Figure Lengend Snippet: SIS3-VAP–DAC nanoemulsion enhances in vitro NK-92 cell-mediated cytotoxicity of osteosarcoma in the presence of TGF-β. Fold-change in MG63-GFP cell count measured by IncuCyte over 48 h following co-incubation with irradiated NK-92 cells (E:T 5:1) in media supplemented with 5 ng/mL TGF-β and 50% (v/v) NE1 (blank), NE2 (SIS3), NE3 (VAP–DAC), or NE4 (SIS3-VAP–DAC) nanoemulsions.

Article Snippet: Recombinant human (rh)IL-2 and rhIL-15 were obtained from the Biological Resources Branch (National Cancer Institute, Frederick, MD) and recombinant murine IL-15 was purchased from R&D Systems (Minneapolis, MN). hSAEC primary small airway epithelial cells (Cat #PCS-301-010), NK-92 human NK cell lymphoma (Cat #CRL-2407), MG63 human osteosarcoma (Cat #CRL-1427), and K7M2 murine osteosarcoma (Cat #CRL-2836) cell lines were all obtained from ATCC (Manassas, VA).

Techniques: In Vitro, Cell Characterization, Incubation, Irradiation

Journal: Frontiers in Immunology

Article Title: Development and evaluation of an inhalable nanoemulsion system for enhancing NK cell function against osteosarcoma pulmonary metastases

doi: 10.3389/fimmu.2026.1772375

Figure Lengend Snippet: Combination SIS3-VAP–DAC nanoemulsion with adoptive NK-92 cell therapy reduces pulmonary osteosarcoma metastases in vivo without systemic toxicity. (A) Representative bioluminescence images of mice from each treatment group at week 1 of dosing. (B) Chest-region bioluminescence ROI values at week 1 across treatment groups. Box and whisker plots display quantitative ROI measurements from the chest area of individual mice, with the middle line indicating the median value. Data represent pulmonary tumor burden after one week of treatment. Whiskers represent data within 1.5 times the interquartile range. (C) Representative bioluminescence images of mice from each treatment group at week 2 of dosing. (D) Chest-region bioluminescence ROI values at week 2 across treatment groups. Box and whisker plots display quantitative ROI measurements from the chest area of individual mice, with the middle line indicating the median value. Data represent pulmonary tumor burden after two weeks of treatment. Whiskers represent data within 1.5 times the interquartile range. (E) Representative bioluminescence images of mice from each treatment group at week 3 of dosing. (F) Chest-region bioluminescence ROI values at week 3 across treatment groups. Box and whisker plots display quantitative ROI measurements from the chest area of individual mice, with the middle line indicating the median value. Data represent pulmonary tumor burden after three weeks of treatment. Whiskers represent data within 1.5 times the interquartile range. At weeks 2 and 3 (D, F) , asterisks denote statistically significant differences between NE7 and the control group by Kruskal–Wallis non−parametric analysis of variance followed by Dunn’s post hoc test (*: p < 0.05, Bonferroni corrected). No significant differences were detected at week 1 (B) . (G) Percentage change in mean body weight over time for control and nanoemulsion-treated mice (NE5-NE7) following MG63 cancer cell injection. Error bars represent ± SD of the percentage changes from baseline at each timepoint. (H) Summed clinical scores in control and nanoemulsion-treated mice (NE5-NE7) over 49 days after MG63 cancer cell injection. Points at zero indicate no observable adverse clinical signs.

Article Snippet: Recombinant human (rh)IL-2 and rhIL-15 were obtained from the Biological Resources Branch (National Cancer Institute, Frederick, MD) and recombinant murine IL-15 was purchased from R&D Systems (Minneapolis, MN). hSAEC primary small airway epithelial cells (Cat #PCS-301-010), NK-92 human NK cell lymphoma (Cat #CRL-2407), MG63 human osteosarcoma (Cat #CRL-1427), and K7M2 murine osteosarcoma (Cat #CRL-2836) cell lines were all obtained from ATCC (Manassas, VA).

Techniques: In Vivo, Whisker Assay, Control, Injection

Journal: International Journal of Cancer

Article Title: Targeting class I histone deacetylases by the novel small molecule inhibitor 4 SC ‐202 blocks oncogenic hedgehog‐ GLI signaling and overcomes smoothened inhibitor resistance

doi: 10.1002/ijc.31117

Figure Lengend Snippet: Inhibition of HH/GLI signaling by 4SC‐202. ( a ) Quantification of Hh/Gli signal strength in response to 4SC‐202 treatment using NIH/3T3 Hh reporter cells containing an 8x‐Gli binding site driving luciferase expression in response to Hh pathway activation. Hh/Gli luciferase reporter cells were stimulated with 1 μg/ml of recombinant Shh and treated with the respective 4SC‐202 concentrations. Luciferase activity and cell viability were measured after 24 hr of treatment. RLU: relative light units. ( b and c ) qPCR and Western blot analysis of HDAC1/2/3 mRNA levels in Daoy ( b ) and protein expression in HH‐responsive Daoy and NIH/3T3 cells ( c ). ( d and e ) qPCR analysis of GLI1 ( d ) and HHIP ( e ) mRNA expression in SAG‐stimulated Daoy cells exposed to increasing concentrations of 4SC‐202. GLI1 and HHIP mRNA levels in SAG‐treated Daoy cells without 4SC‐202 were set to 100%. ( f ) Western blot analysis of Daoy cells treated with respective concentrations of vismodegib or 4SC‐202. ( g ) Relative densitometric quantification of protein levels shown in ( f ). ( h ) Proliferation analysis of SAG‐stimulated Daoy cells in response to 4SC‐202 treatment at the concentrations indicated. β‐Tubulin or total ERK1/2 served as loading controls for Western blots.

Article Snippet: To study human HH/GLI signaling, we applied Daoy medulloblastoma cells (ATCC HTB‐186) responsive to chemical and genetic pathway activation.

Techniques: Inhibition, Binding Assay, Luciferase, Expressing, Activation Assay, Recombinant, Activity Assay, Western Blot

Journal: International Journal of Cancer

Article Title: Targeting class I histone deacetylases by the novel small molecule inhibitor 4 SC ‐202 blocks oncogenic hedgehog‐ GLI signaling and overcomes smoothened inhibitor resistance

doi: 10.1002/ijc.31117

Figure Lengend Snippet: 4SC‐202 inhibits HH/GLI signaling by targeting class I HDACs rather than LSD1. ( a ) Summary of inhibitors used for chemical perturbations and their molecular targets. ( b ) qPCR analysis of GLI1 mRNA expression in SAG‐stimulated Daoy cells in response to increasing concentrations of the LSD1 blocker OG‐L002. ( c ) GLI1 mRNA expression analysis in SAG‐induced or control‐treated Daoy cells transfected with control siRNA (siControl) or siRNA against LSD1 (siLSD1). ( d , e ) qPCR analysis of GLI1 ( d ) and HHIP ( e ) mRNA expression in SAG‐stimulated Daoy cells in response to the HDAC1/2/3 inhibitor entinostat. ( f ) GLI1 mRNA expression in Daoy cells treated with the FDA‐approved pan‐HDAC inhibitor SAHA (vorinostat).

Article Snippet: To study human HH/GLI signaling, we applied Daoy medulloblastoma cells (ATCC HTB‐186) responsive to chemical and genetic pathway activation.

Techniques: Expressing, Control, Transfection

Journal: International Journal of Cancer

Article Title: Targeting class I histone deacetylases by the novel small molecule inhibitor 4 SC ‐202 blocks oncogenic hedgehog‐ GLI signaling and overcomes smoothened inhibitor resistance

doi: 10.1002/ijc.31117

Figure Lengend Snippet: 4SC‐202 inhibits HH/GLI signaling in SMOi‐resistant cancer cells. ( a ) Western blot analysis of GLI1 expression in cells with lentiviral shRNA‐mediated knockdown of SUFU (shSUFU) or control knockdown (shControl). Beta Actin served as loading control. ( b ) qPCR analysis of GLI1 mRNA expression in SUFU knockdown (shSUFU) and control cells (shControl) transduced with lentiviral nontargeting scrambled shRNA. ( c and d ) qPCR analysis of GLI1 ( c ) and HHIP mRNA expression ( d ) as readout for HH/GLI signaling activity in SUFU‐depleted SMOi‐resistant cells showing resistance to vismodegib but sensitivity to 4SC‐202 treatment. ( e ) Western blot analysis of SUFU‐depleted Daoy cells treated with vismodegib or 4SC‐202 at the concentrations indicated. β‐Tubulin served as loading control. Vismodegib was unable to reduce GLI1 protein levels, while 4SC‐202 treatment effectively abolished GLI1 protein expression. ( f ) Relative densitometric quantification of GLI1/β‐Tubulin protein levels shown in ( e ). ( g ) ChIP analysis of MYC‐tagged GLI1 binding to the GLI target promoter PTCH in response to control or 4SC‐202 treatment. Enrichment of GLI1 bound promoter DNA was measured by qPCR. IgG isotype antibody was used as control. ( h ) Murine BCC cells were subcutaneously injected into dorsal flanks of 12 NSG mice. 4SC‐202 was administered by oral gavage at 80 mg/kg/day. The tumor volume at day 0 (i.e., start of drug treatment (arrow)) was set to 100%. ( i ) Western blot analysis of solvent (allografts #1–#4) and 4SC‐202 treated (allografts #5–#8) BCC lysates probed for proliferation‐cell‐nuclear‐antigen (Pcna) and Ccnd1 expression. Erk1/2 expression served as loading control. ( j ) Analysis of in vitro cell proliferation of murine BCC cells in response to 4SC‐202 and entinostat treatment. ChIP: chromatin immunoprecipitation. ** p < 0.01, *** p < 0.001.

Article Snippet: To study human HH/GLI signaling, we applied Daoy medulloblastoma cells (ATCC HTB‐186) responsive to chemical and genetic pathway activation.

Techniques: Western Blot, Expressing, shRNA, Knockdown, Control, Transduction, Activity Assay, Binding Assay, Injection, Solvent, In Vitro, Chromatin Immunoprecipitation

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: Expression of CDK7 in osteosarcoma cell lines, osteosarcoma patient fresh tissues, and osteosarcoma tissue microarray (TMA). (a) Expression levels of CDK7 protein in osteosarcoma cell lines (U2OS, KHOS, 143B, Saos-2, MG63, and MNNG/HOS) were higher than the expression of CDK7 in the normal osteoblast cell line (HOB-c, NHOst) as measured by Western blot. (b) Densitometry quantification of the Western blots of CDK7 from (a), presented as relative to tubulin expression. The data represent the mean ± SE of the experiment carried out in triplicate. (c) CDK7 expression in seven human osteosarcoma fresh tissues measured by Western blot. (d) Densitometry quantification of the Western blots of CDK7 from (c), presented as relative to tubulin expression. The data represent the mean ± SE of the experiment carried out in triplicate. (e) Representative images of different immunohistochemistry staining intensities of CDK7 and HE staining in an osteosarcoma TMA are shown in osteosarcoma tissues. Based on the CDK7 staining intensity within the tumor samples, the staining patterns were divided into six groups: no positive staining (0); <10% positive cells (1+); 10–25% positive cells (2+); 26–50% positive cells (3+); 51–75% positive cells (4+); >75% positive cells (5+). (Original magnification, 400×; scale bar, 50 µm). (f) Tumors with the staining score of ⩽2+ were defined as the low CDK7 expression group, ⩾3+ were defined as the high CDK7 expression group. Pie chart representing relative frequency of different CDK7 expression levels in osteosarcoma TMA. CKD7, cyclin-dependent kinase 7.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Microarray, Western Blot, Immunohistochemistry, Staining

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: Effects of the CDK7 inhibitor BS-181 on the activity of CDK7 and cell growth in osteosarcoma cell lines. BS-181, at the indicated concentrations, inhibited osteosarcoma cell proliferation in (a) cell growth and proliferation of KHOS, and (b) U2OS cell lines, which was determined by MTT assays after treatment with BS-181 for 6 days. The data represent the mean ± SE of two experiments carried out in triplicate. (c) Microscopy images of morphologic changes and a reduction in cell number after 72 h of BS-181 treatment (scale bar, 100 µm). (d) The expression of proteins involved in the CDK7-signaling pathway in the KHOS osteosarcoma cell line was examined by Western blot after 48 h of BS-181 treatment. (e) Semiquantitative analysis of (d) densitometry relative to tubulin. The data represent the mean ± SE of the experiment carried out in triplicate. (f) The expression of proteins involved in the CDK7-signaling pathway in the U2OS osteosarcoma cell line was examined by Western blot after 48 h of BS-181 treatment. (g) Semiquantitative analysis of (f) densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate. CKD7, cyclin-dependent kinase 7; RNAPII, RNA polymerase II.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activity Assay, Microscopy, Expressing, Western Blot

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: CDK7 inhibition reduced osteosarcoma cell clonogenicity in vitro and decreased the spheroid diameter of osteosarcoma cell lines in a three-dimensional (3D) cell culture. (a) Representative results of colony formation in KHOS and U2OS. The numbers of colonies and their sizes were markedly decreased in cells treated with BS-181. (b, c) Representative images of KHOS and U2OS were measured after 10 µM of BS-181 treatment in a 3D cell culture. Spheroid formation of (b) KHOS and (c) U2OS were significantly smaller than untreated cells at all observation points. Cell fluorescence images of spheroid formation were taken after 12 days of cultivation (scale bar, 100 µm). (d, e) The average relative spheroid diameter of (b) KHOS and (c) U2OS treated with CDK7 inhibitor compared with untreated cells at an observation point of 12 days. The data are mean ± SE of the two experiments carried out in triplicate (** indicates p < 0.001). CKD7, cyclin-dependent kinase 7.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Inhibition, In Vitro, Cell Culture, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas

doi: 10.1136/jitc-2020-001355

Figure Lengend Snippet: Combined IL-15 stimulation and TIGIT blockade augments killing of sarcomas in vitro when target expression of TIGIT ligands is high. Analysis of the TCGA-SARC database by Kaplan-Meier analysis shows a significant survival advantage in tumors with a (A) high NK signature (p=0.0003), (B) high CD8 signature (p=0.03), but independent of (C) TIGIT expression (p=0.17). (D) TIGIT expression within STS tumors has a strong positive correlation with NK signature (r=0.82, p<0.0001) and CD8 signature (r=0.90, p<0.0001). (E) Low expression of the primary TIGIT ligand CD155 was associated with improved overall survival by Kaplan-Meier analysis of the TCGA-SARC database (p=0.04). (F) Expression of TIGIT ligand CD155 on sarcoma cell lines A673 (red) and SK-LMS (blue) compared with FMO staining. (G) Increased cytotoxicity of CFSE-labeled target cells A673 (left) and SK-LMS (right) following 4-hour incubation with in vitro IL-15-preactivated PBMCs from a patient with sarcoma in the presence anti-TIGIT 10 ug/mL (red) compared with IgG control 10 ug/mL (blue) at low and high E:T ratios. Increased expression of degranulation marker CD107a on NK cells from IL-15-preactivated PBMCs following 4-hour incubation with sarcoma cell lines shown by (H) representative flow cytometry against A673 cells, and (I) quantified per cent expression against A673 and SK-LMS cell lines in a 5:1 E:T ratio. These experiments were repeated independently in two patients with STS with representative data shown. P value determined using Student’s t-test using n=3–4 technical replicates. FMO, Fluorescence Minus One; IL, interleukin; PBMCs, peripheral blood mononuclear cells; STS, soft tissue sarcoma; TCGA, The Cancer Genome Atlas.

Article Snippet: Human sarcoma cell lines A673 (#CRL-1598, Ewing’s sarcoma) and SK-LMS (#HTB-88, leiomyosarcoma) were obtained from ATCC (Manassas, Virginia, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Nu-Serum and 1% penicillin-streptomycin.

Techniques: In Vitro, Expressing, Staining, Labeling, Incubation, Control, Marker, Flow Cytometry, Fluorescence

Journal: Cancer Research

Article Title: Antibody-Dependent Cell Cytotoxicity Synapses Form in Mice during Tumor-Specific Antibody Immunotherapy

doi: 10.1158/0008-5472.can-10-4222

Figure Lengend Snippet: Figure 1. Antigen-specific binding of the Chi-Tn mAb to the plasma membrane of ovarian and breast cancers. A, a representative human serous ovarian adenocarcinoma and a ductal infiltrating breast cancer were labelled with the biotinylated Chi-Tn mAb (brown areas). Magnifications 20 and 40 are shown. B, cells from a representative ovarian cancer ascitis of patient were labelled with anti-Epcam-FITC, anti-CD45- PerCP-Cy5.5, biotinylated-Chi-Tn mAb (dashed line) or biotinylated- Chi-Tn mAb preincubated with GalNAc (continuous line), then with streptavidine-PE. Epcam þ CD45-epithelial cells were gated in DAPI-negative living cells, and the Chi-Tn (PE) mean fluorescence intensity (MFI) was determined (MFI Chi-Tn þ GalNAc: 239; MFI Chi-Tn: 1693). C, TA3Ha, Jurkat, and SKBR3 cells were incubated with rituximab (thin line), trastuzumab (dashed line) or Chi-Tn mAb (bold line), and GaH-Fc-PE. DOHH2 cells were labelled using biotinylated rituximab (thin line) or biotinylated trastuzumab (dashed line) or biotinylated Chi-Tn (bold line), and strepavidin-PE. D, Chi- Tn mAb was preincubated with soluble GalNAc (thin lines) or GlcNAc (control sugar, dashed lines). Jurkat and TA3Ha cells were incubated with each mixture or with the Chi-Tn mAb alone (dotted line), then with GaH-Fc- PE. Gray histograms: cells incubated with GaH-Fc-PE alone.

Article Snippet: DOHH2 cells (human follicular B lymphoma) were purchased from DSMZ and cultured in RPMI 1640 containing 5% human AB serum.

Techniques: Binding Assay, Clinical Proteomics, Membrane, Fluorescence, Incubation, Control

Journal: Cancer Research

Article Title: Antibody-Dependent Cell Cytotoxicity Synapses Form in Mice during Tumor-Specific Antibody Immunotherapy

doi: 10.1158/0008-5472.can-10-4222

Figure Lengend Snippet: Figure 2. The Chi-Tn mAb does not affect TA3Ha cell viability or growth in vitro. A, DOHH2, Jurkat, and TA3Ha cells were cultured for 24 hours with Chi-Tn mAb (squares) or rituximab (circles), in the presence (open symbols) or absence (filled symbols) of GaH- Fc antiserum. Percentages of PI- positive dead cells were determined by flow cytometry. B, SKBR3 or TA3Ha cells were incubated with trastuzumab (black bars) or Chi-Tn mAb (white bars), or doxorubicin (positive control, 1 mg/mL, gray bar) for 6 days. Cell proliferation was expressed as a percentage of inhibition compared with untreated cells.

Article Snippet: DOHH2 cells (human follicular B lymphoma) were purchased from DSMZ and cultured in RPMI 1640 containing 5% human AB serum.

Techniques: In Vitro, Cell Culture, Flow Cytometry, Incubation, Positive Control, Inhibition

Journal: Cellular and Molecular Immunology

Article Title: A modified HLA-A*0201-restricted CTL epitope from human oncoprotein (hPEBP4) induces more efficient antitumor responses

doi: 10.1038/cmi.2017.155

Figure Lengend Snippet: Immunogenicity of the P40–48 peptide in HLA-A2.1/Kb Tg mice. HLA-A2.1/Kb Tg mice (seven mice per group) were immunized with phosphate-buffered saline (PBS) or P40–48-pulsed DCs, and pAd-GFP- or pAd-hPEBP4-transduced DCs as described in the Materials and methods. Splenocytes from immunized mice were restimulated in vitro with P40–48 for 7 days. SSp-1 was used as a nonspecific control peptide. (a) IFN-γ-positive SFCs/106 splenocytes in ELISPOT assay. (b) IFN-γ-staining of CD8+ T cells (flow cytometry data are representative of three independent experiments). (c and d) Cytotoxicity measured by standard 51Cr-release assays at the indicated E:T ratio. (c) P40–48-pulsed T2 and (d) MCF-7 (hPEBP4+/HLA-A2.1+) cells were used as peptide-specific targets, while (c) SSp-1-pulsed T2 cells or T2 cells alone and (d) A549 (hPEBP4−/HLA-A2.1+) or Daudi (hPEBP4−/HLA-A2.1−) cells were used as control targets. Data are presented as the mean±s.e.m. of three independent experiments. **P<0.01; *P<0.05. CTL, cytotoxic T lymphocyte; DC, dendritic cell; ELISPOT, enzyme-linked immunospot; E:T, effector:target; hPEBP4, human phosphatidylethanolamine-binding protein 4; IFN, interferon; PE, phycoerythrin; SFC, spot-forming cell; Tg, transgenic.

Article Snippet: Human transporter associated with antigen processing (TAP)-deficient T2 cells (expressing HLA-A2.1 molecules), human breast adenocarcinoma MCF-7 (hPEBP4 + /HLA-A2.1 + ), human Burkitt’s lymphoma Daudi (hPEBP4 + / HLA-A2.1 − ) and human lung carcinoma A549 (hPEBP4 − / HLA-A2.1 + ) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Immunopeptidomics, Saline, In Vitro, Control, Enzyme-linked Immunospot, Staining, Flow Cytometry, Binding Assay, Transgenic Assay

Journal: Cellular and Molecular Immunology

Article Title: A modified HLA-A*0201-restricted CTL epitope from human oncoprotein (hPEBP4) induces more efficient antitumor responses

doi: 10.1038/cmi.2017.155

Figure Lengend Snippet: Cytotoxicity of CD8+ CTLs raised by in vitro stimulation of PBLs of HLA-A*0201-positive breast carcinoma patients. CTLs were induced with autologous P40–48-pulsed DCs from PBLs of hPEBP4+/HLA-A2.1+ breast carcinoma patients. On day 7 after the third stimulation, CD8+ T cells were enriched by positive selection using magnetic immunobeads for analysis. (a) IFN-γ-positive SFCs/106 CD8+ T cells detected by cytokine-specific ELISPOT. (b) IFN-γ staining of CD8+ T cells (flow cytometry data are representative of three independent experiments). (c) Cytotoxicity measured by 51Cr-release assays at the indicated E:T ratio. P40–48-pulsed T2 cells and MCF-7 cells (hPEBP4+/HLA-A2.1+) were used as peptide-specific targets, while SSp-1-pulsed T2 cells or T2 cells alone and A549 (hPEBP4−/HLA-A2.1+) or Daudi (hPEBP4−/HLA-A2.1−) cells were used as controls. Data are presented as the mean±s.e.m. of three independent experiments. **P<0.01; *P<0.05. CTL, cytotoxic T lymphocyte; DC, dendritic cell; ELISPOT, enzyme-linked immunospot; E:T, effector:target; hPEBP4, human phosphatidylethanolamine-binding protein 4; IFN, interferon; PBL, peripheral blood lymphocyte; PE, phycoerythrin; SFC, spot-forming cell; Tg, transgenic.

Article Snippet: Human transporter associated with antigen processing (TAP)-deficient T2 cells (expressing HLA-A2.1 molecules), human breast adenocarcinoma MCF-7 (hPEBP4 + /HLA-A2.1 + ), human Burkitt’s lymphoma Daudi (hPEBP4 + / HLA-A2.1 − ) and human lung carcinoma A549 (hPEBP4 − / HLA-A2.1 + ) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Selection, Enzyme-linked Immunospot, Staining, Flow Cytometry, Binding Assay, Transgenic Assay

Journal: Cellular and Molecular Immunology

Article Title: A modified HLA-A*0201-restricted CTL epitope from human oncoprotein (hPEBP4) induces more efficient antitumor responses

doi: 10.1038/cmi.2017.155

Figure Lengend Snippet: 1Y3W6L, 1F7F, 1Y6V and 1F analogs induce more efficient hPEBP4-specific CTLs than the native P40–48 epitope in HLA-A2.1/Kb Tg mice. Splenocytes from HLA-A2.1/Kb Tg mice (seven mice per group) immunized with native P40–48- or analog-pulsed DCs were restimulated in vitro with P40–48 for 7 days. Ex vivo IFN-γ ELISA (a) and IFN-γ ELISPOT (b) of splenocytes. Data are presented as the mean±s.e.m. of three independent experiments. **P<0.01, *P<0.05. Cytotoxicity against P40–48-pulsed T2 cells examined by (c) granzyme B ELISPOT and (d) 51Cr-release assay. In (c), the number of granzyme B-positive SFCs from 105 splenocytes is calculated. In (d), P40–48-pulsed T2, full-length hPEBP4-transfected MAD-MB-468 (MAD-MB-468-hPEBP4, HLA-A2.1+) and MCF-7 (hPEBP4+/HLA-A2.1+) cells were used as hPEBP4-specific HLA-A2.1-restricted target cells. SSp-1 peptide-pulsed T2, non-pulsed T2, Daudi (hPEBP4+, HLA-A2.1−) and MAD-MB-468 (hPEBP4−/HLA-A2.1+) cells were used as controls. The cytotoxicity of various CTLs against the corresponding target cells was tested at a 10:1 E:T ratio in the granzyme B ELISPOT assay and at a 50:1 E:T ratio in the 51Cr-release assay. Data are presented as the mean±s.e.m. of three independent experiments. **P<0.01, *P<0.05. All results. CTL, cytotoxic T lymphocyte; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot; E:T, effector:target; hPEBP4, human phosphatidylethanolamine-binding protein 4; IFN, interferon; SFC, spot-forming cell; siRNA, small interfering RNA.

Article Snippet: Human transporter associated with antigen processing (TAP)-deficient T2 cells (expressing HLA-A2.1 molecules), human breast adenocarcinoma MCF-7 (hPEBP4 + /HLA-A2.1 + ), human Burkitt’s lymphoma Daudi (hPEBP4 + / HLA-A2.1 − ) and human lung carcinoma A549 (hPEBP4 − / HLA-A2.1 + ) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Ex Vivo, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Release Assay, Transfection, Binding Assay, Small Interfering RNA

Journal: Cellular and Molecular Immunology

Article Title: A modified HLA-A*0201-restricted CTL epitope from human oncoprotein (hPEBP4) induces more efficient antitumor responses

doi: 10.1038/cmi.2017.155

Figure Lengend Snippet: 1Y3W6L and 1Y6V analogs induced enhanced CD8+ T-cell responses in PBLs from HLA-A*0201-positive breast cancer patients. PBLs from HLA-A*0201-positive breast cancer patients were stimulated with 1Y3W6L, 1F7F, 1Y6V, 1F or native P40–48 peptides three times at weekly intervals as described in the Materials and methods. On day 7 after the third stimulation, CD8+ T cells were enriched by positive selection using magnetic immunobeads for analysis (a–d). The CTLs in PBLs were then evaluated for IFN-γ release (a and b), granzyme B release (c) and 51Cr-release (d) against T2 cells prepulsed with P40–48 or hPEBP4-expressing HLA-A*0201-positive tumor cells. The number of IFN-γ-positive and granzyme B-positive SFCs was determined from 106 (b) or 105 (c) CD8+ T lymphocytes, respectively. (d) P40–48-pulsed T2, MAD-MB-468-hPEBP4 (hPEBP4+/HLA-A2.1+) and MCF-7 (hPEBP4+/HLA-A2.1+) cells were used as the hPEBP4-specific HLA-A2.1-restricted target cells. SSp-1 peptide-pulsed T2, non-pulsed T2, Daudi (hPEBP4+/HLA-A2.1−) and MAD-MB-468 (hPEBP4−/HLA-A2.1+) cells were used as controls. The cytotoxicity of various CTLs against the corresponding target cells was tested at a 10:1 E:T ratio in the granzyme B ELISPOT assay and at a 50:1 E:T ratio in the 51Cr-release assay. Data are presented as the mean±s.e.m. of three independent experiments. **P<0.01, *P<0.05. CTL, cytotoxic T lymphocyte; DC, dendritic cell; ELISPOT, enzyme-linked immunospot; E:T, effector:target; hPEBP4, human phosphatidylethanolamine-binding protein 4; IFN, interferon; PBL, peripheral blood lymphocyte; SFC, spot-forming cell; siRNA, small interfering RNA.

Article Snippet: Human transporter associated with antigen processing (TAP)-deficient T2 cells (expressing HLA-A2.1 molecules), human breast adenocarcinoma MCF-7 (hPEBP4 + /HLA-A2.1 + ), human Burkitt’s lymphoma Daudi (hPEBP4 + / HLA-A2.1 − ) and human lung carcinoma A549 (hPEBP4 − / HLA-A2.1 + ) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Selection, Expressing, Enzyme-linked Immunospot, Release Assay, Binding Assay, Small Interfering RNA

Journal: Cancer Research

Article Title: Adenoviral E1A Targets Mdm4 to Stabilize Tumor Suppressor p53

doi: 10.1158/0008-5472.can-04-2419

Figure Lengend Snippet: Fig. 2. E1A stabilizes p53 through Mdm4 independently of p14ARF. A, E1A stabilized p53 in MCF-7 cells. The expressions of Mdm4 and p53 were examined by Western blot analysis of extracts from MCF-7 and MCF/E1A cells. B. The Mdm4 expression construct was transfected into MCF-7 and MCF/E1A cells. After transfection for 20 hours, p53 proteins were measured by Western blot analysis. C. E1A failed to stabilize p53 in the absence of Mdm4. Equal amounts of p53 cDNA (0.5 g) were cotransfected with or without E1A (1.0 g) into p53/ MEF, Mdm2/ plus p53/ double knockout MEF, and Mdm4/ plus p53/ double knockout MEF cells, respectively. Cells were harvested before p53 or E1A-induced apoptotic effect was significant. Cell lysates were prepared after 10 hours, and p53 expression was measured by Western blot analysis. The p53 levels were normalized with actin levels, and the p53 increase folds of Lane 3 versus Lane 2, Lane 6 versus Lane 5, and Lane 9 versus Lane 8 are shown at the bottom.

Article Snippet: Human cell lines MCF-7 (breast cancer, wild-type p53), U2OS (osteosarcoma, wild-type p53), and 293T (expressing wild-type p53 and adenoviral E1A/E1B) were obtained from the American Type Culture Collection (Manassas, VA). p53-null, Mdm2/p53 double null, and Mdm4/p53 double null mouse embryo fibroblast (MEF) cells were generated from knockout mice as described previously (19).

Techniques: Western Blot, Expressing, Construct, Transfection, Double Knockout

Journal: Cancer Research

Article Title: Adenoviral E1A Targets Mdm4 to Stabilize Tumor Suppressor p53

doi: 10.1158/0008-5472.can-04-2419

Figure Lengend Snippet: Fig. 3. E1A forms complex with p53 in the presence of Mdm4, facilitating Mmd4-p53 binding while inhibiting Mdm4-Mdm2 binding. A, GST pulldown assay was performed using GST-E1A and S35-labeled in vitro transcribed and translated Mdm4 and p53. B, The binding of Mdm4 to p53 in the presence of purified E1A protein in vitro was measured by GST pulldown assay. C, The Mdm4-myc construct was transfected into MCF-7 and MCF/E1A cells. The cells were treated with MG132 (20 mol/L) for 2 hours before the cell lysate was prepared. Mdm4 was immunoprecipitated (IP) by the myc antibody, and the Mdm4-Mdm2 interaction then was examined by immunoblot with Mdm2 antibody. 9083

Article Snippet: Human cell lines MCF-7 (breast cancer, wild-type p53), U2OS (osteosarcoma, wild-type p53), and 293T (expressing wild-type p53 and adenoviral E1A/E1B) were obtained from the American Type Culture Collection (Manassas, VA). p53-null, Mdm2/p53 double null, and Mdm4/p53 double null mouse embryo fibroblast (MEF) cells were generated from knockout mice as described previously (19).

Techniques: Binding Assay, GST Pulldown Assay, Labeling, In Vitro, Purification, Construct, Transfection, Immunoprecipitation, Western Blot

Journal: Cancer Research

Article Title: Adenoviral E1A Targets Mdm4 to Stabilize Tumor Suppressor p53

doi: 10.1158/0008-5472.can-04-2419

Figure Lengend Snippet: Fig. 4. E1A-induced p53 stabilization accompanies accumulation of ubiquitinated p53. A. HA-ubiquitin was cotransfected with 12S E1A into MCF-7 cells. After transfection for 18 hours, cells were treated with or without MG132 (20 mol/L) for 6 hours, and p53 proteins were immunoprecipitated (IP) from the extracts. The ubiquitination (Ub) of p53 was shown. B. MCF-7 and MCF/E1A cells were treated with MG132 (20 mol/L) for 6 hours, and cell lysates then were prepared. The Mdm2-p53 complexes were examined by immunoprecipitation and Western blot analysis.

Article Snippet: Human cell lines MCF-7 (breast cancer, wild-type p53), U2OS (osteosarcoma, wild-type p53), and 293T (expressing wild-type p53 and adenoviral E1A/E1B) were obtained from the American Type Culture Collection (Manassas, VA). p53-null, Mdm2/p53 double null, and Mdm4/p53 double null mouse embryo fibroblast (MEF) cells were generated from knockout mice as described previously (19).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot

Journal: Cancer Research

Article Title: Adenoviral E1A Targets Mdm4 to Stabilize Tumor Suppressor p53

doi: 10.1158/0008-5472.can-04-2419

Figure Lengend Snippet: Fig. 5. E1A enhances the nuclear retention of p53. A. MCF-7 and MCF/E1A cells were treated with or without MG132 (20 mol/L) for 6 hours. The levels of p53 proteins were analyzed by Western blot analysis and normalized with actin levels. B. Nuclear and cytoplasmic extracts were prepared from MCF-7 and MCF/E1A cells treated with or without MG132 (20 mol/L) for 6 hours. The nuclear distribution of p53 or Mdm4 was analyzed by Western blot analysis. C, p53 (top) time course change of the cytoplasmic p53 level in MCF and MCF/E1A cells after inhibition of protein degradation. The cytoplasmic fraction of MCF-7 and MCF/E1A cell lysates was prepared after the treatment of MG132 (20 mol/L) for different times; the p53, E1A, and -actin proteins were detected by Western blot analysis. Bottom, the changed fold of cytoplasmic p53 level in top. The intensity of bands in the immunoblot is quantitated using image analysis software Quantity One 4.3.0 (Bio-Rad, Hercules, CA), and the ratio of p53 level at each time point to that at time 0 is shown. 9084

Article Snippet: Human cell lines MCF-7 (breast cancer, wild-type p53), U2OS (osteosarcoma, wild-type p53), and 293T (expressing wild-type p53 and adenoviral E1A/E1B) were obtained from the American Type Culture Collection (Manassas, VA). p53-null, Mdm2/p53 double null, and Mdm4/p53 double null mouse embryo fibroblast (MEF) cells were generated from knockout mice as described previously (19).

Techniques: Western Blot, Inhibition, Software

Journal: Frontiers in Pharmacology

Article Title: Fraxinellone Has Anticancer Activity by Inducing Osteosarcoma Cell Apoptosis via Promoting Excessive Autophagy Flux

doi: 10.3389/fphar.2021.653212

Figure Lengend Snippet: FRA inhibited osteosarcoma cell proliferation in vitro . (A) Chemical structure of fraxinellone (FRA). (B) Human osteosarcoma cell lines (HOS & MG63) were treated with different concentrations of FRA for 24 and 48 h before CCK8 assays were utilized. (C) Compared with control group, FRA significantly suppresses colony formation in HOS and MG63 cell lines. (D) Number of clones in plates were analyzed. These data were detected by a microplate reader and a microscope, respectively. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the controls.

Article Snippet: The human osteosarcoma cell lines MNNG/HOS (HOS) (CRL-1547TM, ATCC) and MG63 (CRL-1427TM, ATCC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: In Vitro, Control, Clone Assay, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Fraxinellone Has Anticancer Activity by Inducing Osteosarcoma Cell Apoptosis via Promoting Excessive Autophagy Flux

doi: 10.3389/fphar.2021.653212

Figure Lengend Snippet: FRA inhibits osteosarcoma cell migration in vitro . (A) The HOS and MG63 osteosarcoma cell lines were incubated with various doses of FRA (0; 40 μM; 80 μM) for 12 h, and photographs taken at 0 and 12 h were statistically analyzed by Image J software. The results of wound-healing assay showed that FRA significantly blocked the cell recolonization in the wound area in the HOS and MG63 cell lines. (C) In the transwell assay under the same conditions, the number of cells in of the FRA treatment group was significantly lower than that of the control group, and the number of cells in the high dose was less than that in the low dose. Percentages of wound area (B) and number of clones (D) in the lower membrane were counted. Error bar = mean ± SEM of at least triplicate experiments. Magnification, ×40 (A) , ×100 (C) . Scale bar, 500 μm (A) , 200 μm (C) . * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001.

Article Snippet: The human osteosarcoma cell lines MNNG/HOS (HOS) (CRL-1547TM, ATCC) and MG63 (CRL-1427TM, ATCC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: Migration, In Vitro, Incubation, Software, Wound Healing Assay, Transwell Assay, Control, Clone Assay, Membrane

Journal: Frontiers in Pharmacology

Article Title: Fraxinellone Has Anticancer Activity by Inducing Osteosarcoma Cell Apoptosis via Promoting Excessive Autophagy Flux

doi: 10.3389/fphar.2021.653212

Figure Lengend Snippet: FRA induces autophagy and apoptosis of osteosarcoma cells in vitro . (A) Cells treated with different conditions were stained with Hoechst 33342, and results showed that the percentage of apoptotic cells in the FRA group was higher than in the control group. The highlight dots mean apoptotic cells. (B) After staining with an Annexin FITC/PI kit, the treated cells were tested by flow cytometry. The results indicated that the percent of total apoptotic cells significantly increased in the FRA-treated group in a dose manner. (C) The expression of the following apoptosis-related proteins was determined by Western blot analysis: Bcl-2,cleaved-caspase 3, caspase-3, cleaved−caspase 7, and caspase 7. (D) The expression of those autophagy-related proteins: Beclin-1, ATG5, LC3B and P62. (E) Cells treated with different conditions were stained with MDC, and results showed that the number of autophagosome in the FRA group was higher than in the control group. The green dots mean autophagosome. (F) The HOS and MG63 osteosarcoma cell lines were incubated with FRA (80 μM) or control for 24 h. The expression level of the Beclin-1 and P62 were significantly increased after FRA treatment detected via immunofluorescence assays. Each assay was repeated three times. Magnification, ×200 (A) , ×1000 (D) , ×200 (F) . Scale bar, 100 μm (A) , 20 μm (E) , 100 μm (F) . Error bar = mean ± SD of at least triplicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Article Snippet: The human osteosarcoma cell lines MNNG/HOS (HOS) (CRL-1547TM, ATCC) and MG63 (CRL-1427TM, ATCC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: In Vitro, Staining, Control, Flow Cytometry, Expressing, Western Blot, Incubation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: 89 Zr-CD25 IgG uptake in mouse cells in vitro and in mouse lymphoma in vivo . (A) CD25 expression (left) and anti-mouse 89 Zr-CD25 IgG binding (right) in mouse thymus Treg lymphocytes and EL4 mouse lymphoma cells. Cell uptake was compared by using equivalent amounts of cells. Blocking was done by adding 0.5 μM of unlabeled anti-CD25 IgG. (B) Representative coronal (left) and transaxial (right) PET images at 5 days of EL4 tumor-bearing immunocompetent C57/Bl6 mice administered with anti-mouse 89 Zr-CD25 IgG alone (top) or co-injected and 0.8 mg of unlabeled anti-CD25 IgG for blocking (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 6 per group) N.S., not significant.

Article Snippet: Human anaplastic large cell lymphoma SUDHL-1 cells and EL4 mouse T lymphoma cells were from the American Type Culture Collection and was tested negative for mycoplasma and authenticated by the Institutional Research Service.

Techniques: In Vitro, In Vivo, Expressing, Binding Assay, Blocking Assay, Injection